Complete human day 14 post-implantation embryo models from naive ES cells

The ability to study human post-implantation development remains limited owing to ethical and technical challenges associated with intrauterine development after implantation1. Embryo-like models with spatially organized morphogenesis and structure of all defining embryonic and extra-embryonic tissues of the post-implantation human conceptus (that is, the embryonic disc, the bilaminar disc, the yolk sac, the chorionic sac and the surrounding trophoblast layer) remain lacking1,2. Mouse naive embryonic stem cells have recently been shown to give rise to embryonic and extra-embryonic stem cells capable of self-assembling into post-gastrulation structured stem-cell-based embryo models with spatially organized morphogenesis (called SEMs)3. Here we extend those findings to humans using only genetically unmodified human naive embryonic stem cells (cultured in human enhanced naive stem cell medium conditions)4. Such human fully integrated and complete SEMs recapitulate the organization of nearly all known lineages and compartments of post-implantation human embryos, including the epiblast, the hypoblast, the extra-embryonic mesoderm and the trophoblast layer surrounding the latter compartments. These human complete SEMs demonstrated developmental growth dynamics that resemble key hallmarks of post-implantation stage embryogenesis up to 13–14 days after fertilization (Carnegie stage 6a). These include embryonic disc and bilaminar disc formation, epiblast lumenogenesis, polarized amniogenesis, anterior–posterior symmetry breaking, primordial germ-cell specification, polarized yolk sac with visceral and parietal endoderm formation, extra-embryonic mesoderm expansion that defines a chorionic cavity and a connecting stalk, and a trophoblast-surrounding compartment demonstrating syncytium and lacunae formation. This SEM platform will probably enable the experimental investigation of previously inaccessible windows of human early post implantation up to peri-gastrulation development.

The ability to capture human pluripotent stem cells (ESCs) in different developmental states in culture 57,58 has opened the possibility of investigating early human embryogenesis using 3D stem-cell derived embryo models.By controlling cellular composition and differentiation conditions, the recently introduced elegant models such as gastruloids, blastoids, axioloids, or amniotic sac embryoids [59][60][61][62][63][64][65][66][67][68][69][70] recapitulate some aspects of early mammalian development in a modular manner.Among them, Blastocystlike structures (blastoids) are the only currently available human integrated (i.e., comprised of all embryonic and extra-embryonic lineages) model of the human pre-implantation embryo, containing epiblast, primitive endoderm (PrE), and trophectoderm (TE), that can be made with high efficiencies nearing 70%.So far, the attempts to advance the blastoids towards post-implantation stages through in vitro culture did not progress beyond what has been achieved with natural human blastocysts 6 .Similarly, mouse blastoids do not develop into bona fide post-implantation embryos even when transferred in utero 71 .It is possible that technical improvements in the future might enable further development of blastoids through and beyond gastrulation.

Supplementary Discussion
In this study, we found conditions that allowed naïve (HENSM) human ESCs to form the three extra-embryonic lineages and embryonic epiblast that together are required to assemble a post implantation embryonic structure without transgene induction.These cells assembled into a complex structure that highly resembles the structural and sequential patterning of all embryonic and extraembryonic lineages of the postimplantation human embryo up to initiation of gastrulation.These structures are integrated complete SEMs that mimic human embryo development ex utero up to an equivalent of 14 dpc.The human postimplantation integrated SEM generation protocol devised herein reveals the remarkable self-organizing ability of naïve ESCs and provide a window into hereto near impossible to study period of human development.
The fact that human (and mouse) SEM formation bypasses the blastocyst-like stage and that the ex vivo protocols for growing natural human blastocysts into the authentic 14 dpf stage are still lacking, makes donated fetal materials from 7-14 dpf the only relevant control for benchmarking the SEM describe herein.
Obviously, obtaining new 7-14 dpf human embryo samples is not a viable option for both technical and ethical reasons.However, this limitation is partially mitigated by the available Carnegie and other embryo atlas collections 20,39 , as well as the ability to compare human development to NHP embryo models and datasets.However, due to the lack of perfect reference controls, we cannot and do not claim that human SEMs generated herein are identical to natural day 14 human embryos, and it is most likely they are not.
We also cannot exclude that some cell types might be missing from the SEMs generated herein or that some of the extra-embryonic-like lineages being primed by protocols devised herein might contain other lineages that are normally not found in the early-post-implantation natural human embryo.
Regarding the variability in developmental stage that can be observed between day 6-8, it could reflect natural variability that could occur during natural development, such as that seen in mouse in utero development where 0.5-1 days variability can be observed.Alternatively, it is possible that this variability results from inherent heterogeneity in the starting induced cell populations made to generate the aggregates or reflect suboptimal protocol development.Developing new conditions that can maintain TE-like state and stable PrE-and ExEM-like cells might contribute to enhancing SEM formation efficiency and reducing variability.
It is possible that the high ectopic expression produced by the PiggyBac system of GATA3 or CDX2, in addition to the endogenous levels induced by the media, leads to unfavorably high levels of such factors which might derail the differentiation outcome and TE/TSC-like properties.Using roller culture after day 6 with the same media composition did not yield a better outcome than orbital shaking placed in static incubators (Supplementary Fig. 10e) 3,43 .The latter is consistent with our results in mouse embryos, where the roller culture platform was essential for late gastrulation and organogenesis stages.
Naïve pluripotent stem cell growth conditions typically utilize FGF/MEK signaling inhibition (including HENSM used herein), which leads to the loss of imprinting after extended passaging 72,73 , perturbing the developmental potential of such cells and thus might partially underlie the low efficiency yield and SEM quality.This risk may possibly be mitigated in the future by using naïve conditions with titrating down the concentration of FGF/MEK pathway inhibitors or using alternative naïve conditions that do not target this pathway 4,72 , which is of future scientific importance to explore and optimize.
It is plausible that upon further experimentation and mechanistic understanding of selforganization, the efficiency and variability in SEM formation can be improved in the future.Devising serum free defined conditions for all different stages of this protocol is also of technical importance.It is also likely that alternative ex utero culture platforms, aggregation strategies, or growth conditions will yield similar or enhanced results relative to the ones reported herein with human SEMs.Developing a variety of PSC lines each carrying multiple endogenous fluorescent reporters for different lineage markers is likely to be useful to assess of SEMs with live-cell imaging technologies and add great ease to select those that can be of high-quality for further expansion ex utero (rather than relying only on aggregate morphology).
Finally, incorporating multi markers staining or FISHs technologies (like HCR -multiplexed, quantitative, high-resolution RNA fluorescence in situ hybridization (RNA-FISH)), might enable evaluating simultaneous presence of multiple compartments and cell types in each SEM.

Supplementary Figure 1 .
Cell lines and strategies to obtain and define PrE/ExEM-like cells from human naïve ESCs.a, scheme of the donor plasmid vector used for genomic integration of the DOX-inducible iGATA4 or iGATA6 overexpression in human ESCs.b, representative immunofluorescence images of mono-and polyclonal iGATA4 (left) and iGATA6 (right) WIBR3 hESC (W3) clones, showing uniform expression of GATA4 (green) and GATA6 (green) in response to DOX; nuclei (DAPI, blue).Scale bars, 50 µm.c, control FACS plots of human cardiomyocyte precursor cells (CMPCs, known to express high levels of PDGFRa) unstained (left) or stained with anti-human PDGFR (right).d, FACS plots of unstained (left) and antihuman PDGFRa-stained (right) mouse embryonic fibroblasts (MEFs), validating specificity of the antihuman PDGFRa antibody.e, FACS plots of MEFs either unstained (left) or stained with anti-mouse PDGFRa antibody (right).The staining pattern validates high species-specificity of the used antibodies and the none-to-negligible contribution of MEFs to the PDGFRa+ fraction when confluent human cells, following MEF depletion, were stained with the human-specific anti-PDGFRa antibody.f, Representative qRT-PCR gene expression (normalized by GAPDH and ACTIN) of the endodermal markers for PrE/ExEM-like cell induction in RCL medium starting form naïve (blue) vs primed (red) human ESC.Naïve human ESCs maintained in HENSM (white) were used as a reference control.DEdefinitive endoderm.Values of each sample represent average value of 3 technical replicates per sample, error bars indicate s.d..A single representative experiment out of N=3 biological replicates performed is shown.g (left to right), numbers of genes, total read counts, and percentage of mitochondrial read counts in the scRNA-seq samples of HENSM ESC derived cells induced in BAP(J) or RCL conditions for 3 days.Supplementary Figure 4. Characterizing the capacity of CDX2 overexpression to generate TE/TSClike cells from human naïve ESCs compatible with successful SEM formation.a, scheme of the donor plasmid vector used for genomic integration of the DOX-inducible CDX2 overexpression in human ESCs.b, representative immunofluorescence images of iCDX2 cells, showing CDX2 (green) expression only in response to DOX; nuclei (DAPI, blue).Scale bars, 50 µm.c, representative images of iCDX2 (left) and WT (right) human W3 hESCs in HENSM conditions, showing live fluorescence of tdTomato (red) after transfection with lentiviral particles carrying the fluorophore.Scale bars, 50 µm.d (top), the scheme of the experiment, where naïve ESCs and hypoblast/ExEM-like induced cells were aggregated with iCDX2 cells, induced by DOX for 48h in three different media, PALY (N2B27 supplemented with PD0325901, A83-01, hLIF, and Y-27632), TSCm 13 , and BAP(J) (DMEM/F12 based medium with ALK4/5/7 inhibitor A83-01, FGF2 inhibitor PD0325901, and BMP4 substituted with JAK inhibitor I after 24h).d (bottom), representative immunofluorescence images showing no surrounding trophoblast in aggregates with iCDX2 cells, regardless of the media conditions; epiblast (OCT4, cyan), hypoblast (GATA4, SOX17, yellow), trophoblast (CK7, SDC1, magenta), nuclei (DAPI, blue).Scale bars (from left to right), 100 µm, 50 µm, 100 µm.e (top), the scheme of the experiment, in which naïve ESCs and PrE/ExEM-like induced cells were aggregated with WT or iCDX2 tdTomato-labelled nESC induced towards trophectoderm (TE) in PALY media with or without DOX as indicated.e (bottom), representative brightfield and live fluorescence images of day 4 -7 aggregates showing localization of the trophoblast.Scale bar, 200 µm.f, representative phase contrast microscopy images showing iCDX2 cells induced for 72h in different media (HENSM, BAP(J), TSCm) with or without DOX, showing reduced viability upon iCDX2 transgene overexpression.In all conditions, 750,000 iCDX2 cells were seeded in 10 cm Matrigel coated plates, and DOX induction was started 24h after seeding.Scale bars, 100 µm.Supplementary Figure 6.Human naïve ESC derived TSC lines do not sort adequately in human SEM protocol.a, representative immunofluorescence images validating correct expression of TSC marker genes in colonies of a WIBR3 (W3) naïve hESC-derived TSC line (termed nTSC).CDX2, GATA3, TFAP2C, CK7 (all in green); DAPI (blue).Scale bar, 100 µm.b, scheme for aggregation protocol of naïve pluripotent stem cells (nESCs) in HENSM media, naïve-derived trophoblast stem cells (nTSCs), and nESCs induced in RCL towards PrE/ExEM-like cells for 3 days.c (left), representative immunofluorescence images showing rare expression of CK7, GATA3, and SDC1 (all in red) trophoblast markers in the aggregates.nuclei (DAPI, white); scale bar, 500 µm.c (right), zoom into several aggregates with CK7 expression; scale bar, 100 µm.d, representative immunofluorescence image from (upper left panel in (c), showing Epilike cells (OCT4, cyan) and PrE-like cells (SOX17, yellow) with CK7 (red); nuclei (DAPI, white).Bottom, zoom images of the outlined regions are shown.Although some aggregates express lineage markers, they do not organize into embryoid-like structures and are not uniformly surrounded by the trophoblast.Scale bar, 500 µm (top); bottom, 100 µm.Supplementary Introduction Our understanding of human early post-implantation development has been limited due to difficulties in obtaining relevant embryo samples from these early stages of human gestation, and technical and ethical challenges for in vitro development of donated blastocysts towards the post-implantation stages while preserving the in vivo complexity and correct spatial morphogenesis of both embryonic and extra embryonic compartments.Most of our knowledge on critical early post-implantation stages of human embryo development has been gained solely from histological and anatomical descriptions in embryological collections, and the experimental models for mechanistic research are lacking.Despite the important recent advances 18 in culturing the in vitro attached human embryos beyond implantation, these culture systems are restricted in sample number and, overall, do not support normal development of embryos beyond the initiation of early epiblast lumenogenesis 15 .